Thyroid tumors are the most common endocrine malignancy. It has been estimated that up to 90% of the autopsies performed in this country reveal the presence of slow-growing thyroid tumors. While the prognosis for patients with well-differentiated follicular and papillary thyroid tumors is good, anaplastic thyroid tumors are rapidly fatal. Activating Ras mutations are particularly prevalent in human thyroid tumors. Ras mutations are found in benign adenomas and at a higher frequency in follicular and anaplastic carcinomas. Mutations in B-Raf, a downstream Ras effector, are the most frequent mutational event in papillary thyroid tumors. These observations support roles for Ras in the initiation and progression of thyroid tumors. A large proportion of papillary thyroid tumors exhibit amplification and rearrangement of the PKCepsilon gene, leading to the expression of an N-terminal fragment of PKCepsilon structurally similar to the V1 domain, a peptide that selectively inhibits PKCepsilon translocation. Interestingly, most papillary thyroid tumors exhibit decreased expression of PKCepsilon. Moreover, expression of the RET/PTC oncogene induced the selective translocation, followed by downregulation of PKCepsilon. PKCalpha expression is increased in follicular thyroid tumors, tumors that also harbor Ras mutations. It is our hypothesis that individual PKC isozymes play essential roles in the initiation and maintenance of thyroid cell transformation by Ras. Our preliminary data demonstrate that PKCdelta selectively reproduces the acute effects of oncogenic Ras on aberrant cell cycle progression and apoptosis; that PKCepsilon is required for Ras-induced morphological changes; that PKCs mimic the inhibitory effects of Ras on thyroid differentiation; and that Ras-transformed thyroid cells exhibit alterations in PKC expression and activity. The proposed studies investigate the roles of individual PKC isozymes in the initiation and maintenance of Ras transformation in rat thyroid cells. This will be accomplished using highly specific molecular reagents including adenoviruses for PKC isozymes, selective PKC peptide activators and inhibitors and RNA interference. This analysis will provide novel insight into the molecular mechanisms through which Ras dysregulates thyroid cell proliferation, differentiation and survival, and may give rise to the development of new strategies to selectively impair tumor cell proliferation and/or reactivate differentiated gene expression.